This application is to request a MERIT Award extension. Our progress validated our hypothesis that globin gene expression and heme biosynthesis are interlinked through GATA-1-dependent mechanisms. Aim 1. To distinguish between models for how heme amplifies GATA-1 activity to coordinate hemoglobin biosynthesis and erythroid cell development/function. We will test whether heme permits or enhances the GATA-1-dependent subnuclear transition that expels target loci from the nuclear periphery (Model 1) or enhances GATA-1 activity subsequent to the transition (Model 2). If heme enhances the GATA-1-dependent subnuclear transition of Bach1-sensitive genes, we will analyze the relationship between Bach1 and other factors/coregulators that drive locus relocalization. As the relationship between subnuclear transitions and chromatin looping remains elusive, we will determine whether heme and Bach1 regulate GATA-1-mediated looping. If heme enhances activation subsequent to the transition, we will dissect late mechanistic steps. Aim 2. To assemble activation and repression matrices and use these unique resources to elucidate how the GATA-1/heme circuit establishes a critical sector of the erythroid cell transcriptome. We hypothesize that GATA-1 target gene cohorts requiring unique ensembles of factors (including heme) and coregulators share common mechanisms/pathways. We assembled first-gen. matrices illustrating relationships between target gene expression and factor/coregulator requirements. Considerably expanded second-gen. matrices will be developed. Unraveling the mechanisms/pathways will yield vital insights into hemoglobin synthesis and erythroid cell development/function. Aim 3. To use a synthetic biology approach involving cis-element engineering to ascertain how GATA-1-binding cis-elements control heme biosynthesis and erythroid biology. Using CRISPR/Cas9, we will rewire the cis-element circuitry controlling heme biosynthesis to determine why the Alas2 intron1 GATA-1-binding cis-element is much more important than the intron8 GATA-1-binding cis-element. We will test the hypothesis that the difference reflects intrinsic differences between the elements, or distinct flanking sequences render the elements differentially active at endogenous loci. We will generate G1E-ER-GATA-1 cells in which the cis-elements are swapped to determine if they retain or adopt new attributes at the ectopic chromosomal site. Concepts will be validated in primary erythroblasts and in vivo. These studies will establish rules governing cis-element function in erythroid cells, which will inform GATA factor-dependent mechanisms, biology, and pathologies. RELEVANCE (See instructions): The proposed studies shall provide fundamental insights into mechanisms underlying disorders of hemoglobin synthesis, including thalassemias and anemias and diseases associated with aberrant heme biosynthesis, including porphyrias. Moreover, the work shall provide a conceptual framework for the design and implementation of translational and therapeutic strategies for these disorders.